There is a continuing need to cultivate lymphoid cells which produce useful biologically active substances such as lymphokines (for example, interferons and interleukins) and monoclonal antibodies (hereinafter also briefly referred to as MoAbs). For the cultivation of lymphoid cell strains, culture media containing about 10% of sera are mainly used as with other cells. In particular, fetal calf serum (FCS)-containing culture media have usually been employed. Serum-free culture media has also been employed, but such media are generally poor in cell proliferation, particularly when compared to the serum-containing culture media. For this reason, it is necessary to increase the seeding density on cell passage when serum-free media is employed. In order to improve proliferation in serum-free culture media, some groups have attempted to obtain cell strains improved in proliferative activity by the method of cell-adaptation to the serum-free culture media or the method of inducing mutations. However, with respect to lymphoid cell lines such as hybridomas, there has been no improvement in the proliferative activity by using recombinant technology.
In recent years, various factors have been isolated and their genes have been elucidated with the advance of investigations on cell growth factors. Fibroblast growth factors (FGFs), one kind of the above-mentioned cell growth factors, are cell growth factors discovered from the bovine pituitary glands by D. Gospodarowicz [Nature 249, 123 (1974)]. There are two types of FGF, basic FGF (bFGF) which is basic in its isoelectric point and acidic FGF (aFGF) which is acidic in its isoelectric point. The amino acid sequences of both bFGF and aFGF have been elucidated [Bovine bFGF: Proc. Natl. Acad. Sci. USA 82, 6507-6511 (1985); Human bFGF: The EMBO Journal 5, 2523-2528 (1986); Human and bovine bFGF: Biochem. Biophys. Res. Commun. 138, 611-617 (1986)]. FGFs are cell growth factors exhibiting proliferation promoting activity in almost all mesoderm-derived cells such as adrenal cell Yl [Endocrinology 97, 120 (1975)], myoblasts [J. Cell Biol. 70, 395 (1976)], chondrocytes [J. Cell. Physiol. 91, 977 ( 1977)] and vascular endothelial cells [Proc. Natl. Acad. Sci. U.S.A. 73, 4120 (1976)], as well as to fibroblasts [J. Cell Biol. 66, 451 (1975)]. However, it is known that FGFs do not generally exhibit proliferation promoting activity in epithelial cells [Saibo Seicho Inshi (Growth Factors), page 32, edited by The Japanese Tissue Culture Association, Asakura Shoten, 1984]. The present inventors have discovered that biologically active substances can be efficiently produced from lymphoid cells such as hybridomas by cultivating the lymphoid cells in FGF-containing culture media. And there is a report that FGFs increase the proliferative response of B cells activated by mitogen to low molecular weight B cell growth factor [Cellular Immunology 122, 424 (1989)]. However, until the present invention, introduction of FGF gene into lymphoid cells to improve the proliferative activity of cells has not been reported at all.
As noted above, the aminoacid and nucleotide sequences of both aFGF and bFGF have already been determined, which sequences reveal that both FGFs are synthesized at first as peptides each having 155 amino acid residues and that no signal peptide sequences for secretion are observed in their nucleotide sequences [J. A. Abraham et al., Science 233, 545-548 (1986); and M. Jaye et al., Science 233, 541- 545 (1986)].
When useful substances are produced by cultivating animal cells, various problems are pointed out about the use of the serum culture media. Namely, the sera vary in quality among lots, and have the danger of infection with mycoplasma and viruses. Further, there is a fear of exerting adverse effects on the quality of the products produced because of the complicated purification process.
The serum-free culture media is therefore advantageously used, but is generally found to be poor in cell proliferation. For this reason, the productivity of such useful substances is diminished in many cases. Under such conditions, where there is poor cell proliferation, the cells are typically required to be inoculated at a high cell density (for example, in the serum-free culture of human-human hybridomas producing human MoAbs, a cell density of about 1.times.10.sup.5 cells/ml is necessary). In large-scale cultivation on an industrial scale, therefore, the complexity of increased cell passage cycles arises, and the danger of contamination with mycoplasma and viruses is also increased. Further, no techniques for cloning cells by limiting dilution culture methods using serum-free culture media have been established yet.
The present inventors considered that such problems could be solved if a mutant strain exhibiting high proliferative activity, even in serum-free culture media, could be obtained. Several attempts were made to improve the cells proliferative activity by conventional methods such as mutations. However, satisfactory results were not obtained. Even if a desired mutant strain is obtained, that method is time-consuming and cannot be said to be a sure method.
In recent years, expression vectors using animal cells as host cells have been developed. The present invention was completed by improving proliferative activity of hybridomas using genetic engineering techniques, thus solving the above-mentioned problems.